which spontaneously develop skin melanomas, l l l THROMBOSIS AND HEMOSTASIS

significantly prolonged survival in these mice. In

particular, reduced VEGF amounts in tumors Comment on Prakash et al, page 3164
and reduced tumor cell proliferation were
affected by tinzaparin treatment, which likely
reflect a reduced angiogenesis and cancer
Fibronectin: extra domain
progression.
Taken together, the work of Bauer et al1
brings extra risk?
-----------------------------------------------------------------------------------------------------
provides an important contribution to
our understanding of cancer-induced Yiming Wang1,2 and Heyu Ni1,2 1
ST. MICHAEL’S HOSPITAL; 2CANADIAN BLOOD SERVICES

prothrombic activity with regard to

endothelial activation and VWF fiber In this issue of Blood, Prakash et al provide compelling in vitro and in vivo
formation in the lumen of cancer-associated evidence for a novel role of cellular fibronectin (cFn) containing extra domain A
vasculature. Although this work convincingly (Fn-EDA1) in arterial thrombosis.1

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shows the relevance of VWF fibers formation
and associated platelet aggregation in primary
tumors of cancer patients, the fundamental F ibronectins (Fn’s) are large dimeric
multidomain glycoproteins consisting of
two 230- to 250-kDa monomers linked by
Using intravital microscopy models, the
roles of both pFn and cFn in thrombosis have
been established in the past decade.4-7 pFn is
question as whether VWF fiber formation occurs
during metastatic initiation requires further disulfide bonds at their C terminus. Each Fn synthesized in the hepatocytes and accounts for
investigation. Along these lines, the current monomer contains collagen and fibrin binding almost all of the Fn’s in the normal blood
findings show that endothelial activation is sites in the N terminus and an Arg-Gly-Asp plasma. The depletion of pFn in mice resulted
a critical requirement for successful metastasis. (RGD) integrin binding motif. The alternative in a decrease of thrombus growth and stability.4
A better understanding of molecular events splicing of Fn pre-messenger RNA (pre- It was later found that pFn supports platelet
leading to VWF fiber deposition and subsequent mRNA) generates several Fn variants, which aggregation and thrombosis only in the
microthrombus formation could allow the can be grouped into 2 main categories: plasma presence of fibrin, and the pFn-fibrin complex
designing of new therapy approaches. fibronectin (pFn) and cFn. In contrast to pFn, may be a prerequisite for its prothrombotic
Conflict-of-interest disclosure: The author cFn contains either EDA or EDB or both.2 activities.5,6 In the absence of fibrin (eg, at the
declares no competing financial interests. n Fn’s are widely distributed in extracellular apical surface of the growing thrombi), pFn
matrix and blood circulation. They are inhibits platelet aggregation, thus preventing
required for embryogenesis, and are important excessive thrombosis and rescuing downstream
REFERENCES
for wound healing, infection, and malignant blood supply.6 pFn also supports hemostasis
1. Bauer AT, Suckau J, Frank K, et al. von Willebrand
factor fibers promote cancer-associated platelet aggre-
transformation.3 through depositing on the injured vessel wall
gation in malignant melanoma of mice and humans. Blood.
2015;125(20):3153-3163.
2. Varki A. Trousseau’s syndrome: multiple definitions
and multiple mechanisms. Blood. 2007;110(6):1723-1729.
3. Rickles FR. Mechanisms of cancer-induced throm-
bosis in cancer. Pathophysiol Haemost Thromb. 2006;35(1-2):
103-110.
4. Wagner DD, Frenette PS. The vessel wall and its
interactions. Blood. 2008;111(11):5271-5281.
5. Brill A, Fuchs TA, Chauhan AK, et al. von Willebrand
factor-mediated platelet adhesion is critical for deep vein
thrombosis in mouse models. Blood. 2011;117(4):1400-1407.
6. Lip GY, Chin BS, Blann AD. Cancer and the
prothrombotic state. Lancet Oncol. 2002;3(1):27-34.
7. Kerk N, Strozyk EA, Pöppelmann B, Schneider SW.
The mechanism of melanoma-associated thrombin activity
and von Willebrand factor release from endothelial cells.
J Invest Dermatol. 2010;130(9):2259-2268.
8. Falanga A, Marchetti M. Heparin in tumor
progression and metastatic dissemination. Semin Thromb
Hemost. 2007;33(7):688-694.
9. Varki NM, Varki A. Heparin inhibition of selectin-
mediated interactions during the hematogenous phase of
carcinoma metastasis: rationale for clinical studies in
humans. Semin Thromb Hemost. 2002;28(1):53-66.
10. Borsig L. Antimetastatic activities of heparins and
modified heparins. Experimental evidence. Thromb Res.
2010;125(Suppl 2):S66-S71. Fn-EDA1 interacts with platelet TLR4 to promote thrombosis, possibly through the platelet NF-kB signaling pathway.
Platelet intracellular Fn-EDA1 is packaged from megakaryocytes and possibly also internalized via aIIbb3 integrins,
which are released upon activation from platelet a-granules. Fn-EDA1 may incorporate into fibrin clots, deposit on
© 2015 by The American Society of Hematology vessel wall, and induce NET formation.

BLOOD, 14 MAY 2015 x VOLUME 125, NUMBER 20 3043

and strengthening the fibrin clot.6 cFn is
synthesized by many cell types and joins the
extracellular matrix locally. Although only
minute amounts of cFn can be detected in
blood plasma of healthy populations, plasma
concentration of the EDA containing cFn,
Fn-EDA1, is elevated in disease conditions
such as atherosclerosis, ischemic stroke, and
diabetes. To address the role of Fn-EDA1,
Chauhan and colleagues previously
studied thrombosis in a mouse model
that constitutively expresses Fn-EDA1
but not pFn in the liver.7 Strikingly, despite
a 70% to 80% decrease in total plasma Fn
level, significantly enhanced thrombosis was
observed in these mice as compared with
wild-type mice with normal circulating pFn
concentrations. This finding reveals an
important fact: the extra domain in Fn-EDA1
does bring a higher thrombotic risk than
pFn (ie, Fn’s lack EDA). However, the
mechanism for this prothrombotic function
was previously unclear.
Here, Prakash and colleagues have
addressed whether the observed
prothrombotic effect of Fn-EDA1 can be
mediated through its direct interaction with
platelets.1 Using the FeCl3-induced carotid
injury intravital microscopy model, they
found that Fn-EDA1/1 mice exhibited
significantly enhanced thrombosis compared
with Fn-EDA2/2 mice. However, this
prothrombotic phenotype was abolished in
the absence of Toll-like receptor 4 (TLR4).
They further demonstrated that Fn-EDA1
targets TLR4 in hematopoietic lineage cells
following bone marrow transplants with
or without TLR4 expression (EDA1/1/
TLR41/1 or EDA1/1/TLR42/2) into
Fn-EDA1/1 mice. Finally, after depletion of
endogenous platelets in Fn-EDA1/1 mice,
mice transfused with EDA1/1/TLR41/1
but not EDA1/1/TLR42/2 platelets showed
enhanced thrombosis, thus localizing the
downstream of Fn-EDA1 prothrombotic
function to platelet TLR4. In addition,
activation of a downstream nuclear factor kB
(NF-kB) signaling pathway within the platelet
was found following TLR4 activation by
Fn-EDA1. This informative study reveals
a novel Fn-EDA1 and platelet TLR4
interaction that contributes to thrombosis,
3044
suggesting a possible link between elevated
Fn-EDA1, innate immunity, and thrombotic
risk.
Is TLR4 the only explanation?8 One
interesting finding of this article is that the
prothrombotic effect of Fn-EDA1 in in
vivo thrombosis models seems more striking
than in vitro platelet aggregation assay.1
Could interaction between Fn-EDA1 and
fibrin also contribute to the enhanced
thrombosis? Fn’s can be covalently linked with
the fibrin network, and pFn was found to
strengthen the fibrin clot.6 Because cFn
possesses a greater tendency for polymerization
than pFn, it is possible that Fn-EDA1 may
enhance the clot stiffness even further. Bacterial
lipopolysaccharide (LPS), a TLR4 ligand, has
been reported to interact with platelet TLR4 to
induce neutrophil extracellular trap (NET)
formation.9 Could Fn-EDA1 induce NET
formation through TLR4? As the link
between NETs and thrombosis has been
established recently, it is tempting to
contemplate that Fn-EDA1 may also
enhance thrombosis by promoting NET
formation. These possibilities are especially
appealing for the study of deep vein
thrombosis, inflammation, and innate
immunity, in which both fibrin and NETs
play a vital role (see figure).
Platelets contain both pFn (;80%
of platelet Fn’s) and cFn. pFn is internalized
through platelet surface aIIbb3 integrin,10
whereas cFn is synthesized in megakaryocytes
and packaged in platelets. Although present
at relatively small amounts, these Fn’s can
be released from a-granules upon platelet
activation and boost the Fn concentration at the
site of thrombosis (see figure). With an increase
in Fn-EDA1 in blood plasma, the platelet
Fn-EDA1 likely also increases via aIIbb3
integrin internalization. Could the platelet
endogenous Fn-EDA1 also contribute to
thrombosis? Another interesting fact is that
platelet aggregation and thrombus formation
persist in the absence of both fibrinogen and
von Willebrand factor (VWF), 2 factors
previously considered essential for thrombosis.
The platelet content of Fn in fibrinogen- and
VWF-deficient animals is threefold to fivefold
higher. Although pFn has been subsequently
ruled out as the factor to mediate fibrinogen-
and VWF-independent platelet aggregation,
the prothrombotic Fn-EDA1 remains
a possible candidate.5
In summary, the study from Prakash
and colleagues is another solid step toward
understanding how extra domains regulate Fn
function. Fn’s, either cFn or pFn, change their
circulating concentration under many disease
conditions. The low level of ED-containing
cFn in normal blood circulation may be a result
of natural selection to reduce thrombotic
risk. Further investigations of circulating
Fn-EDA1 in disease conditions may establish
it as a potential therapeutic target for
Downloaded from http://ashpublications.org/blood/article-pdf/125/20/3043/1385753/3043.pdf by guest on 22 January 2021
thrombotic disorders.
Conflict-of-interest disclosure: The authors
declare no competing financial interests. n
REFERENCES
1. Prakash P, Kulkarni PP, Lentz SR, Chauhan AK.
Cellular fibronectin containing extra domain A promotes
arterial thrombosis in mice through platelet Toll-like
receptor 4. Blood. 2015;125(20):3164-3172.
2. Cho J, Mosher DF. Role of fibronectin assembly in
platelet thrombus formation. J Thromb Haemost. 2006;4(7):
1461-1469.
3. Hynes RO. Fibronectins. New York, NY: Springer
Verlag; 1990.
4. Ni H, Yuen PS, Papalia JM, et al. Plasma fibronectin
promotes thrombus growth and stability in injured
arterioles. Proc Natl Acad Sci USA. 2003;100(5):
2415-2419.
5. Reheman A, Yang H, Zhu G, et al. Plasma fibronectin
depletion enhances platelet aggregation and thrombus
formation in mice lacking fibrinogen and von Willebrand
factor. Blood. 2009;113(8):1809-1817.
6. Wang Y, Reheman A, Spring CM, et al. Plasma
fibronectin supports hemostasis and regulates thrombosis.
J Clin Invest. 2014;124(10):4281-4293.
7. Chauhan AK, Kisucka J, Cozzi MR, et al.
Prothrombotic effects of fibronectin isoforms containing
the EDA domain. Arterioscler Thromb Vasc Biol. 2008;
28(2):296-301.
8. Maurer E, Receveur N, Bourdon C, et al. Integrin a5b1
and glycoprotein VI play important roles in mediating
activation and aggregation of platelets adhering to fibrillar
fibronectin. In: Proceedings from the XXIV Congress of
International Society on Thrombosis and Haemostasis
(ISTH); June 29-July 4, 2013; Amsterdam, The
Netherlands. Abstract OC 87.3.
9. Clark SR, Ma AC, Tavener SA, et al. Platelet TLR4
activates neutrophil extracellular traps to ensnare bacteria
in septic blood. Nat Med. 2007;13(4):463-469.
10. Ni H, Papalia JM, Degen JL, Wagner DD. Control of
thrombus embolization and fibronectin internalization by
integrin aIIbb3 engagement of the fibrinogen g chain.
Blood. 2003;102(10):3609-3614.
© 2015 by The American Society of Hematology
BLOOD, 14 MAY 2015 x VOLUME 125, NUMBER 20